FBW7 Mutations Research Articles

Association of patient and disease characteristics with outcomes of regorafenib in patients with metastatic colorectal cancer.

135 Background: Regorafenib (rego) is a multikinase inhibitor approved for use in metastatic colorectal cancer (mCRC). No predictive biomarkers have been identified for rego. We conducted a real-world multi-institutional retrospective study to identify the patient and tumor characteristics associated with outcomes of rego in mCRC patients. Methods: Patients from University of Pittsburgh Medical Center and Montefiore Medical Center who received rego for mCRC were included. Patient characteristics, tumor pathology and molecular profiles, treatment history, and response to rego were collected. Primary and secondary outcomes were best objective response and progression free survival (PFS) on rego, respectively. Univariate and multivariate analyses were conducted using Fisher’s exact test, Mann-Whitney U test, Firth logistic regression and Cox proportional-hazards model. Results: 76 patients were included (median age 64, 42% females, 84% whites). Rego yielded a disease control rate of 32% (including 2 complete responses) and a median PFS of 3.6 months. Tumors with high microsatellite instability (MSI-H) had a significantly lower odds of progression of disease (POD) as best objective response (p = 0.01) and a significantly longer median PFS (p = 0.022) compared with tumors with microsatellite stability (MSS) in multivariable models (Table). 75% of MSI-H mCRC patients had not received immunotherapy (IO) prior to rego. Presence of peritoneal carcinomatosis and older age were associated with higher odds of POD (p = 0.093) and a significantly shorter PFS (p = 0.029), respectively. Compared to Whites, Black patients had a numerically higher odds of POD and shorter PFS. Sex, smoking, alcohol, sidedness of CRC, previous treatments, organs involved, and KRAS mutation were not associated with treatment response or PFS. Conclusions: Our study showed that MSI-H mCRC was associated with significantly better response to rego and PFS, even in the absence of prior IO. Other predicting factors for worse outcomes of rego included older age, and potentially peritoneal carcinomatosis and Black race. None of the mutations was found associated with treatment outcomes, with the limitation of lack of comprehensive molecular testing in many patients. Studies to further examine potential biomarkers such as MCL-1 and FBW7 mutations and correlation to clinical outcomes are ongoing. [Table: see text]

Global and context-specific transcriptional consequences of oncogenic Fbw7 mutations.

The Fbw7 ubiquitin ligase targets many proteins for proteasomal degradation, which include oncogenic transcription factors (TFs) (e.g., c-Myc, c-Jun, and Notch). Fbw7 is a tumor suppressor and tumors often contain mutations in FBXW7, the gene that encodes Fbw7. The complexity of its substrate network has obscured the mechanisms of Fbw7-associated tumorigenesis, yet this understanding is needed for developing therapies. We used an integrated approach employing RNA-Seq and high-resolution mapping (cleavage under target and release using nuclease) of histone modifications and TF occupancy (c-Jun and c-Myc) to examine the combinatorial effects of misregulated Fbw7 substrates in colorectal cancer (CRC) cells with engineered tumor-associated FBXW7 null or missense mutations. Both Fbw7 mutations caused widespread transcriptional changes associated with active chromatin and altered TF occupancy: some were common to both Fbw7 mutant cell lines, whereas others were mutation specific. We identified loci where both Jun and Myc were coregulated by Fbw7, suggesting that substrates may have synergistic effects. One coregulated gene was CIITA, the master regulator of MHC Class II gene expression. Fbw7 loss increased MHC Class II expression and Fbw7 mutations were correlated with increased CIITA expression in TCGA colorectal tumors and cell lines, which may have immunotherapeutic implications for Fbw7-associated cancers. Analogous studies in neural stem cells in which FBXW7 had been acutely deleted closely mirrored the results in CRC cells. Gene set enrichment analyses revealed Fbw7-associated pathways that were conserved across both cell types that may reflect fundamental Fbw7 functions. These analyses provide a framework for understanding normal and neoplastic context-specific Fbw7 functions.

Wingless and Archipelago, a fly E3 ubiquitin ligase and a homolog of human tumor suppressor FBW7, show an antagonistic relationship in wing development

BackgroundArchipelago (Ago) is a Drosophila homolog of mammalian F-box and WD repeat domain-containing 7 (FBW7, also known as FBXW7). In previous studies, FBW7 has been addressed as a tumor suppressor mediating ubiquitin-dependent proteolysis of several oncogenic proteins. Ubiquitination is a type of protein modification that directs protein for degradation as well as sorting. The level of beta-catenin (β-cat), an intracellular signal transducer in Wnt signaling pathway, is reduced upon overexpression of FBW7 in human cancer cell lines. Loss of function mutations in FBW7 and overactive Wnt signaling have been reported to be responsible for human cancers.ResultsWe found that Ago is important for the formation of shafts in chemosensory bristles at wing margin. This loss of shaft phenotype by knockdown of ago was rescued by knockdown of wingless (wg) whereas wing notching phenotype by knockdown of wg was rescued by knockdown of ago, establishing an antagonistic relationship between ago and wg. In line with this finding, knockdown of ago increased the level of Armadillo (Arm), a homolog of β-cat, in Drosophila tissue. Furthermore, knockdown of ago increased the level of Distal-less (Dll) and extracellular Wg in wing discs. In S2 cells, the amount of secreted Wg was increased by knockdown of Ago but decreased by Ago overexpression. Therefore, Ago plays a previously unidentified role in the inhibition of Wg secretion. Ago-overexpressing clones in wing discs exhibited accumulation of Wg in endoplasmic reticulum (ER), suggesting that Ago prevents Wg protein from moving to Golgi from ER.ConclusionsWe concluded that Ago plays dual roles in inhibiting Wg signaling. First, Ago decreases the level of Arm, by which Wg signaling is downregulated in Wg-responding cells. Second, Ago decreases the level of extracellular Wg by inhibiting movement of Wg from ER to Golgi in Wg-producing cells.

Mcl-1 inhibition overcomes intrinsic and acquired regorafenib resistance in colorectal cancer.

Intrinsic and acquired resistance to targeted therapies is a significant clinical problem in cancer. We previously showed that resistance to regorafenib, a multi-kinase inhibitor for treating colorectal cancer (CRC) patients, can be caused by mutations in the tumor suppressor FBW7, which block degradation of the pro-survival Bcl-2 family protein Mcl-1. We tested if Mcl-1 inhibition can be used to develop a precision combination therapy for overcoming regorafenib resistance.Methods: Small-molecule Mcl-1 inhibitors were tested on CRC cells with knock-in (KI) of a non-degradable Mcl-1. Effects of Mcl-1 inhibitors on regorafenib sensitivity were determined in FBW7-mutant and -wild-type (WT) CRC cells and tumors, and in those with acquired regorafenib resistance due to enriched FBW7 mutations. Furthermore, translational potential was explored by establishing and analyzing FBW7-mutant and -WT patient-derived organoid (PDO) and xenograft (PDX) tumor models.Results: We found that highly potent and specific Mcl-1 inhibitors such as S63845 overcame regorafenib resistance by restoring apoptosis in multiple regorafenib-resistant CRC models. Mcl-1 inhibition re-sensitized CRC tumors with intrinsic and acquired regorafenib resistance in vitro and in vivo, including those with FBW7 mutations. Importantly, Mcl-1 inhibition also sensitized FBW7-mutant PDO and PDX models to regorafenib. In contrast, Mcl-1 inhibition had no effect in FBW7-WT CRCs.Conclusions: Our results demonstrate that Mcl-1 inhibitors can overcome intrinsic and acquired regorafenib resistance in CRCs by restoring apoptotic response. FBW7 mutations might be a potential biomarker predicting for response to the regorafenib/Mcl-1 inhibitor combination.

Pan-cancer transcriptional signatures predictive of oncogenic mutations reveal that Fbw7 regulates cancer cell oxidative metabolism

The Fbw7 (F-box/WD repeat-containing protein 7) ubiquitin ligase targets multiple oncoproteins for degradation and is commonly mutated in cancers. Like other pleiotropic tumor suppressors, Fbw7's complex biology has impeded our understanding of how Fbw7 mutations promote tumorigenesis and hindered the development of targeted therapies. To address these needs, we employed a transfer learning approach to derive gene-expression signatures from The Cancer Gene Atlas datasets that predict Fbw7 mutational status across tumor types and identified the pathways enriched within these signatures. Genes involved in mitochondrial function were highly enriched in pan-cancer signatures that predict Fbw7 mutations. Studies in isogenic colorectal cancer cell lines that differed in Fbw7 mutational status confirmed that Fbw7 mutations increase mitochondrial gene expression. Surprisingly, Fbw7 mutations shifted cellular metabolism toward oxidative phosphorylation and caused context-specific metabolic vulnerabilities. Our approach revealed unexpected metabolic reprogramming and possible therapeutic targets in Fbw7-mutant cancers and provides a framework to study other complex, oncogenic mutations.

The deubiquitinase USP9X regulates FBW7 stability and suppresses colorectal cancer.

The tumor suppressor FBW7 targets oncoproteins such as c-MYC for ubiquitylation and is mutated in several human cancers. We noted that in a substantial percentage of colon cancers, FBW7 protein is undetectable despite the presence of FBW7 mRNA. To understand the molecular mechanism of FBW7 regulation in these cancers, we employed proteomics and identified the deubiquitinase (DUB) USP9X as an FBW7 interactor. USP9X antagonized FBW7 ubiquitylation, and Usp9x deletion caused Fbw7 destabilization. Mice lacking Usp9x in the gut showed reduced secretory cell differentiation and increased progenitor proliferation, phenocopying Fbw7 loss. In addition, Usp9x inactivation impaired intestinal regeneration and increased tumor burden in colitis-associated intestinal cancer. c-Myc heterozygosity abrogated increased progenitor proliferation and tumor burden in Usp9x-deficient mice, suggesting that Usp9x suppresses tumor formation by regulating Fbw7 protein stability and thereby reducing c-Myc. Thus, we identify a tumor suppressor mechanism in the mammalian intestine that arises from the posttranslational regulation of FBW7 by USP9X independent of somatic FBW7 mutations.

Abstract 1030: Metabolic reprogramming caused by mutations of the Fbw7 ubiquitin ligase in colorectal cancer

Abstract Fbw7 is an evolutionary conserved F box protein that functions as a substrate adapter for the Skp1-Cullin 1-F box (SCF) E3 ubiquitin ligase complex. Fbw7 is also a tumor suppressor gene that is mutated in diverse human cancers. Many Fbw7 substrates are oncogenic transcription factors, including c-Myc, Notch, and Jun. Cancer-associated Fbw7 mutations diminish its ability to interact with substrates, which results in inappropriate oncoprotein accumulation. One of the significant challenges for understanding the consequences of Fbw7 mutations in tumors is that Fbw7 coordinately regulates approximately two dozen broadly-acting transcription factors. To address this problem, we used novel computational methods to analyze TCGA gene expression datasets to derive transcriptional signatures that could predict a tumor’s Fbw7 mutational status; these signatures were then analyzed for enrichment of genes involved in specific biological processes. We found that genes associated with mitochondrial function were highly predictive of Fbw7 mutations across several tumor types, suggesting that metabolic reprogramming is a crucial oncogenic consequence of Fbw7 mutations. We validated this prediction by creating isogenic colorectal cancer cells with normal or mutant Fbw7 alleles. We found that mitochondrial signature genes are elevated in Fbw7 mutant cells, which also exhibit increased oxidative metabolism. Moreover, metabolomic studies revealed deregulation of other metabolic pathways in Fbw7-mutant cells, including amino acid and nucleotide biosynthesis. These data suggest that metabolic vulnerabilities may represent novel therapeutic targets in Fbw7-associated cancers. Citation Format: Ryan J. Davis, Mehmet Gonen, David Hockenbery, Adam Margolin, Bruce E. Clurman. Metabolic reprogramming caused by mutations of the Fbw7 ubiquitin ligase in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1030. doi:10.1158/1538-7445.AM2017-1030

Abstract 3143: FBW7 mutations mediate resistance of colorectal cancer to targeted therapies by blocking Mcl-1 degradation

Abstract Colorectal cancer (CRC), the second leading cause of cancer-related deaths in the US, has been treated with targeted therapies. However, the mechanisms of differential responses and resistance of CRCs to targeted therapies are not well understood. In this study, we found that genetic alterations of FBW7, an E3 ubiquitin ligase and a tumor suppressor frequently mutated in CRCs, contribute to resistance to targeted therapies. CRC cells containing FBW7 inactivating mutations are insensitive to clinically used multi-kinase inhibitors of RAS/RAF/MEK/ERK signaling, including regorafenib and sorafenib. In contrast, sensitivity to these agents is not affected by oncogenic mutations in KRAS, BRAF, PIK3CA, or p53. These cells are defective in apoptosis due to blocked degradation of Mcl-1, a pro-survival Bcl-2 family protein. Deleting FBW7 in FBW7-wild-type CRC cells abolishes Mcl-1 degradation and recapitulates the in vitro and in vivo drug resistance phenotypes of FBW7-mutant cells. CRC cells selected for regorafenib resistance have progressive enrichment of pre-existing FBW7 hotspot mutations, and are cross-resistant to other targeted drugs that induce Mcl-1 degradation. Furthermore, a selective Mcl-1 inhibitor restores regorafenib sensitivity in CRC cells with intrinsic or acquired resistance. Together, our results demonstrate FBW7 mutational status as a key genetic determinant of CRC response to targeted therapies, and Mcl-1 as an attractive therapeutic target. Citation Format: Jingshan Tong, Shuai Tan, Fangdong Zou, Jian Yu, Lin Zhang. FBW7 mutations mediate resistance of colorectal cancer to targeted therapies by blocking Mcl-1 degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3143. doi:10.1158/1538-7445.AM2017-3143

FBW7 mutations mediate resistance of colorectal cancer to targeted therapies by blocking Mcl-1 degradation.

Colorectal cancer (CRC), the second leading cause of cancer-related deaths in the US, has been treated with targeted therapies. However, the mechanisms of differential responses and resistance of CRCs to targeted therapies are not well understood. In this study, we found that genetic alterations of FBW7, an E3 ubiquitin ligase and a tumor suppressor frequently mutated in CRCs, contribute to resistance to targeted therapies. CRC cells containing FBW7 inactivating mutations are insensitive to clinically used multi-kinase inhibitors of RAS/RAF/MEK/ERK signaling, including regorafenib and sorafenib. In contrast, sensitivity to these agents is not affected by oncogenic mutations in KRAS, BRAF, PIK3CA, or p53. These cells are defective in apoptosis due to blocked degradation of Mcl-1, a pro-survival Bcl-2 family protein. Deleting FBW7 in FBW7-wild-type CRC cells abolishes Mcl-1 degradation and recapitulates the in vitro and in vivo drug resistance phenotypes of FBW7-mutant cells. CRC cells selected for regorafenib resistance have progressive enrichment of pre-existing FBW7 hotspot mutations, and are cross-resistant to other targeted drugs that induce Mcl-1 degradation. Furthermore, a selective Mcl-1 inhibitor restores regorafenib sensitivity in CRC cells with intrinsic or acquired resistance. Together, our results demonstrate FBW7 mutational status as a key genetic determinant of CRC response to targeted therapies, and Mcl-1 as an attractive therapeutic target.

ERK kinase phosphorylates and destabilizes the tumor suppressor FBW7 in pancreatic cancer.

F-box and WD repeat domain-containing 7 (FBW7) is the substrate recognition component of the Skp1-Cul1-F-box (SCF) ubiquitin ligase complex and functions as a major tumor suppressor by targeting various oncoproteins for degradation. Genomic deletion or mutation of FBW7 has frequently been identified in many human cancers but not in pancreatic ductal adenocarcinoma. Thus it is important to know how the tumor suppressive function of FBW7 is impaired in pancreatic cancer. In this study, we first observed that low FBW7 expression correlated significantly with ERK activation in pancreatic cancer clinical samples, primarily due to KRAS mutations in pancreatic cancer. We further showed that ERK directly interacted with FBW7 and phosphorylated FBW7 at Thr205, which sequentially promoted FBW7 ubiquitination and proteasomal degradation. Furthermore, the phospho-deficient T205A FBW7 mutant is resistant to ERK activation and could significantly suppress pancreatic cancer cell proliferation and tumorigenesis. These results collectively demonstrate how the oncogenic KRAS mutation inhibits the tumor suppressor FBW7, thus revealing an important function of KRAS mutations in promoting pancreatic cancer progression.

NOTCH1 Transcriptionally Activates Deptor and Promotes AKT Activation in Acute T-Cell Lymphoblastic Leukemia

Acute T-cell lymphoblastic leukemia (T-ALL) is a malignant disorder of thymocyte progenitors, accounting for 10-15% of pediatric acute lymphoblastic leukemia. The activation of NOTCH1 signaling in T-ALL, by either NOTCH1 (Weng A et al, Science, 2004) or FBW7 (Thompson BJ, J Exp Med, 2007) mutations, is one of the major events that contribute to disease development. DEPTOR emerges as an endogenous mTOR inhibitor, whose expression is low in most cancer cells but surprisingly high in multiple myeloma, resulting in activated AKT (Peterson TR et al, Cell, 2009). However, its implications in other hematological malignancies and the mechanism of regulation remains largely unknown. In this study, we present a novel NOTCH1-mediated transcriptional regulation of DEPTOR in T-ALL and show that DEPTOR is required for T-ALL cell proliferation and survival, and regulates T-ALL cell glucose metabolism via AKT activation.In T-ALL cells, gamma-secretase inhibitor (GSI) treatments resulted in an appreciable decrease of deptor mRNA (Figure 1A) and protein levels (Figure 1B). We then analyzed 174 T-ALL patient specimens (Haferlach J, J Clin Oncol, 2010) and found the expression level of deptor was highly correlated with notch1 and hes1 (Figure 1C). Sequence analyses within the deptor promoter revealed two CSL binding consensus sequences (Figure 1D) and the results from chromatin immunoprecipitation (ChIP) assays indicated a strong physical interaction of ICN1 and the deptor promoter, which was abolished by gamma-secretase inhibition (Figure 1E). Taken together, our data demonstrate that NOTCH1 directly binds to the deptor promoter and activates transcription.To understand its functional role in T-ALL, we depleted DEPTOR in HPB-ALL cells and observed dramatic decrease of cell viability (Figure 2A and 2B) and noticeable enhancement of cell apoptotic death (Figure 2C). Moreover, DEPTOR knockdown significantly reduced glucose uptake and lactate release (Figure 2D), very likely, due to impaired AKT activation reflected by decreased AKT phosphorylation at the Ser473 and Thr308 sites (Figure 2E). These findings provide strong evidence suggesting that high expression of DEPTOR is essential for maintaining T-ALL cell metabolism and survival through promoting AKT activation.Our findings thus establish DEPTOR as a key regulator in mediating the crosstalk between NOTCH1 and AKT signaling pathways, two master oncogenic players in T-ALL. Furthermore, these data provide rationale of developing small molecules or biological agents targeting DEPTOR, in combination with current therapies, for T-ALL treatments. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Fbw7 dimerization determines the specificity and robustness of substrate degradation

The Fbw7 tumor suppressor targets a broad network of proteins for ubiquitylation. Here we show critical functions for Fbw7 dimerization in regulating the specificity and robustness of degradation. Dimerization enables Fbw7 to target substrates through concerted binding to two suboptimal and independent recognition sites. Accordingly, an endogenous dimerization-deficient Fbw7 mutation stabilizes suboptimal substrates. Dimerization increases Fbw7's robustness by preserving its function in the setting of mutations that disable Fbw7 monomers, thereby buffering against pathogenic mutations. Finally, dimerization regulates Fbw7 stability, and this likely involves Fbw7 trans-autoubiquitylation. Our study reveals novel functions of Fbw7 dimerization and an unanticipated complexity in substrate degradation.

Abstract LB-212: Mcl-1 and FBW7 control a dominant survival pathway underlying HDAC and Bcl-2 inhibitor synergy in squamous cell carcinoma.

Abstract Effective targeted therapeutics for squamous cell carcinoma (SCC) are lacking. Here we uncover Mcl-1 as a dominant and tissue-specific survival factor in SCC, providing a roadmap for a new therapeutic approach. Treatment with the HDAC inhibitor vorinostat regulates Bcl-2 family member expression to disable the Mcl-1 axis and thereby induce apoptosis in SCC cells. Although Mcl-1 dominance renders SCC cells resistant to the BH3 mimetic ABT-737, vorinostat primes them for sensitivity to ABT-737 by shuttling Bim from Mcl-1 to Bcl-2/Bcl-xl, resulting in dramatic synergy for this combination and sustained tumor regression in vivo. Moreover, somatic FBW7 mutation in SCC is associated with stabilized Mcl-1 and high Bim levels, resulting in a poor response to standard chemotherapy but a robust response to HDAC inhibitors and enhanced synergy with combination vorinostat/ABT-737. Collectively, our findings provide a biochemical rationale and predictive markers for the application of this therapeutic combination in SCC. Citation Format: Lei He, Kristine Torres-Lockhart, Nicole Forster, Saranya Ramakrishnan, Patricia Greninger, Mathew J. Garnett, Ultan McDermott, S. Michael Rothenberg, Cyril Benes, Leif Ellisen. Mcl-1 and FBW7 control a dominant survival pathway underlying HDAC and Bcl-2 inhibitor synergy in squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-212. doi:10.1158/1538-7445.AM2013-LB-212

Mcl-1 and FBW7 Control a Dominant Survival Pathway Underlying HDAC and Bcl-2 Inhibitor Synergy in Squamous Cell Carcinoma

Effective targeted therapeutics for squamous cell carcinoma (SCC) are lacking. Here, we uncover Mcl-1 as a dominant and tissue-specific survival factor in SCC, providing a roadmap for a new therapeutic approach. Treatment with the histone deacetylase (HDAC) inhibitor vorinostat regulates Bcl-2 family member expression to disable the Mcl-1 axis and thereby induce apoptosis in SCC cells. Although Mcl-1 dominance renders SCC cells resistant to the BH3-mimetic ABT-737, vorinostat primes them for sensitivity to ABT-737 by shuttling Bim from Mcl-1 to Bcl-2/Bcl-xl, resulting in dramatic synergy for this combination and sustained tumor regression in vivo. Moreover, somatic FBW7 mutation in SCC is associated with stabilized Mcl-1 and high Bim levels, resulting in a poor response to standard chemotherapy but a robust response to HDAC inhibitors and enhanced synergy with the combination vorinostat/ABT-737. Collectively, our findings provide a biochemical rationale and predictive markers for the application of this therapeutic combination in SCC.

Collaboration Between RUNX and NOTCH Pathways in T-Cell Acute Lymphoblastic Leukemia

AbstractAbstract 1279T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy characterized by the clonal outgrowth of developmentally arrested T-lymphoid blasts. Notch signaling is activated by mutation of NOTCH1 and/or FBW7 in over half of cases, and ultimately results in increased expression of target genes via the NOTCH/CSL transcriptional complex. Enforced expression of activated NOTCH1 in mouse hematopoietic progenitors leads to the development of clonal T-cell leukemias, suggesting that collaborating mutations are required for establishment and/or propagation of malignant clones. To identify candidate collaborating loci, Beverly and Capobianco performed a retroviral insertional mutagenesis screen in mice expressing a relatively weak activated Notch1 transgene and found recurrent insertions into Ikaros (Ikzf1). These insertions resulted in expression of dominant negative isoforms of Ikaros and thus potentiated Notch signaling since Ikaros and Notch/CSL compete for occupancy at target gene regulatory elements.In an attempt to identify collaborating mutations outside of the Notch pathway, we performed a similar screen, but employed instead a very potent activated NOTCH1 allele (ΔE) in hopes of saturating the Notch signaling pathway. We thus cloned out the insertion sites from 88 primary mouse leukemias generated by transduction of bone marrow with ΔE retrovirus. While recurrent insertions into Ikzf1 were again identified, we also observed frequent insertions into other regions including the Runx3 locus. The Runx3 integrations were tightly clustered in a region 40–60kb upstream of the transcriptional start site, suggesting the retroviral LTR might be inducing an increase in Runx3 expression. A single integration upstream of Runx1 was also identified in a region frequently mutated in similar screens. Of note, analysis of publically available gene expression profile data revealed that RUNX1 and RUNX3 are ubiquitously expressed in patient T-ALL samples.In order to functionally characterize the roles of RUNX1 and RUNX3 in T-ALL, we utilized lentiviral shRNAs to knock down RUNX1 and/or RUNX3 across a broad panel of 26 human T-ALL cell lines. Despite recent studies suggesting RUNX1 may act as a tumor suppressor in T-ALL, we observed the overwhelming majority of cell lines to show substantial growth defects after knock-down of RUNX1/3 as measured by competitive growth assay. These results were confirmed in a subset of cell lines and also in xenograft-expanded primary T-ALL samples by BrdU incorporation/DNA content assays which showed reduced proliferation/G1 cell cycle arrest following RUNX1/3 knock-down. Conversely, overexpression of RUNX3 induced T-ALL cells to proliferate more rapidly and to resist ABT-263-induced apoptosis.To explore potential target genes responsible for these pro-growth/survival effects, we mined available ChIP-Seq data and found NOTCH1/CSL and RUNX1 binding sites to co-localize within IGF1R and IL7R loci at intronic enhancer-like regions with associated H3K4me1>H3K4me3 marks and reduced H3K27me3 marks. Importantly, we show that NOTCH1 and RUNX factors co-regulate surface protein expression of IGF1R and IL7R in a synergistic/additive manner. As we and others have previously demonstrated important roles for both IGF1R and IL7R in T-ALL cell growth and leukemia-initiating activity, these studies reveal a novel collaborative mechanism between NOTCH1 and RUNX proteins in supporting propagation of established T-ALL disease. Disclosures:No relevant conflicts of interest to declare.

Negative Regulation of the Stability and Tumor Suppressor Function of Fbw7 by the Pin1 Prolyl Isomerase

Fbw7 is the substrate recognition component of the Skp1-Cullin-F-box (SCF)-type E3 ligase complex and a well-characterized tumor suppressor that targets numerous oncoproteins for destruction. Genomic deletion or mutation of FBW7 has been frequently found in various types of human cancers; however, little is known about the upstream signaling pathway(s) governing Fbw7 stability and cellular functions. Here we report that Fbw7 protein destruction and tumor suppressor function are negatively regulated by the prolyl isomerase Pin1. Pin1 interacts with Fbw7 in a phoshorylation-dependent manner and promotes Fbw7 self-ubiquitination and protein degradation by disrupting Fbw7 dimerization. Consequently, overexpressing Pin1 reduces Fbw7 abundance and suppresses Fbw7's ability to inhibit proliferation and transformation. By contrast, depletion of Pin1 in cancer cells leads to elevated Fbw7 expression, which subsequently reduces Mcl-1 abundance, sensitizing cancer cells to Taxol. Thus, Pin1-mediated inhibition of Fbw7 contributes to oncogenesis, and Pin1 may be a promising drug target for anticancer therapy.

Abstract 2034: The Fbw7 tumor suppressor: Genetic and biochemical approaches to study a dimeric ubiquitin ligase

Abstract Fbw7, the human ortholog of yeast Cdc4, is the substrate-recognition component of a cullin-RING ubiquitin ligase and regulates a number of oncoprotein substrates including cyclin E, c-Myc, Notch, and c-Jun. A growing number of additional pro-growth and pro-survival factors have been more recently described as Fbw7 targets. Accordingly, Fbw7 is a tumor suppressor and is mutated in approximately ten percent of human malignancies. Its loss leads to chromosomal instability and tumorigenesis. Fbw7 binds its substrates after they become phosphorylated within a consensus sequence called a Cdc4 phospho-degron (CPD). CPD phosphorylation creates a high-affinity binding site for Fbw7, which brings the ligase machinery into proximity with the substrate, leading to ubiquitylation and proteasomal degradation of the substrate. Interestingly, Fbw7 contains a conserved dimerization domain and we can show the protein primarily forms dimers in cells. One Fbw7 dimer contains two substrate-binding interfaces and may make two independent CPD contacts, but the physiologic function of Fbw7 dimerization is still elusive. Using gene-targeting, we studied the effects of ablating Fbw7 dimerization in vivo and found endogenous cyclin E and c-Myc regulation are moderately affected. Using purified Fbw7 monomers or dimers in in vitro ubiquitylation assays, we observed that a dimeric ligase is able to conjugate ubiquitin moieties onto a wider variety of substrate lysine residues than can the monomeric ligase. This is likely due to the greater spatial reach of the dimer. Additionally, preliminary data shows that the dimer may be capable of catalyzing the formation of non-K48-linked polyubiquitin chains, whereas the monomer is not. Cancer-associated mutations in Fbw7 most often target three arginine residues that form a pocket for binding phosphorylated substrate CPDs. Mutation of any of these arginines render Fbw7 unable to bind substrates and properly regulate their abundance. However, these mutations occur typically in only one allele. Dimerization of Fbw7 may explain why such heterozygous mutations are sufficient for tumorigenesis: the mutant form may dominantly inhibit enzyme function of the wild-type allele through dimerization. We have generated hetero-typic Fbw7 dimers containing one wild-type and one mutant protein in each dimer and are currently studying its binding and ubiquitylation capabilities. Given the importance of Fbw7 and its substrates in tumor-progression pathways, the results of this study will further our understanding of human tumor suppression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2034. doi:1538-7445.AM2012-2034

Sensitivity to antitubulin chemotherapeutics is regulated by MCL1 and FBW7

Microtubules have pivotal roles in fundamental cellular processes and are targets of antitubulin chemotherapeutics. Microtubule-targeted agents such as Taxol and vincristine are prescribed widely for various malignancies, including ovarian and breast adenocarcinomas, non-small-cell lung cancer, leukaemias and lymphomas. These agents arrest cells in mitosis and subsequently induce cell death through poorly defined mechanisms. The strategies that resistant tumour cells use to evade death induced by antitubulin agents are also unclear. Here we show that the pro-survival protein MCL1 (ref. 3) is a crucial regulator of apoptosis triggered by antitubulin chemotherapeutics. During mitotic arrest, MCL1 protein levels decline markedly, through a post-translational mechanism, potentiating cell death. Phosphorylation of MCL1 directs its interaction with the tumour-suppressor protein FBW7, which is the substrate-binding component of a ubiquitin ligase complex. The polyubiquitylation of MCL1 then targets it for proteasomal degradation. The degradation of MCL1 was blocked in patient-derived tumour cells that lacked FBW7 or had loss-of-function mutations in FBW7, conferring resistance to antitubulin agents and promoting chemotherapeutic-induced polyploidy. Additionally, primary tumour samples were enriched for FBW7 inactivation and elevated MCL1 levels, underscoring the prominent roles of these proteins in oncogenesis. Our findings suggest that profiling the FBW7 and MCL1 status of tumours, in terms of protein levels, messenger RNA levels and genetic status, could be useful to predict the response of patients to antitubulin chemotherapeutics.

Nucleolar Targeting of the Fbw7 Ubiquitin Ligase by a Pseudosubstrate and Glycogen Synthase Kinase 3

E3 ubiquitin ligases catalyze protein degradation by the ubiquitin-proteasome system, and their activity is tightly controlled. One level of regulation involves subcellular localization, and the Fbw7 tumor suppressor exemplifies this type of control. Fbw7 is the substrate-binding component of an SCF ubiquitin ligase that degrades critical oncoproteins. Alternative splicing produces three Fbw7 protein isoforms that occupy distinct compartments: Fbw7α is nucleoplasmic, Fbw7β is cytoplasmic, and Fbw7γ is nucleolar. We found that cancer-associated Fbw7 mutations that disrupt substrate binding prevent Fbw7γ nucleolar localization, implicating a substrate-like interaction in nucleolar targeting. We identified EBNA1-binding protein 2 (Ebp2) as the critical nucleolar factor that directly mediates Fbw7 nucleolar targeting. Ebp2 binds to Fbw7 like a substrate, and this is mediated by an Ebp2 degron that is phosphorylated by glycogen synthase kinase 3. However, despite these canonical substrate-like interactions, Fbw7 binding is largely uncoupled from Ebp2 turnover in vivo. Ebp2 thus acts like a pseudosubstrate that directly recruits Fbw7 to nucleoli.

Notch3-mediated regulation of MKP-1 levels promotes survival of T acute lymphoblastic leukemia cells

Activation of the Notch pathway occurs commonly in T acute lymphoblastic leukemia (T-ALL) because of mutations in Notch1 or Fbw7 and is involved in the regulation of cell proliferation and survival. Deregulated Notch3 signalling has also been shown to promote leukemogenesis in transgenic mice, but the targets of Notch3 in human T-ALL cells remain poorly characterized. Here, we show that Notch3 controls levels of mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1). In a model of T-ALL cell dormancy, both Notch3 activation and MKP-1 expression were upregulated in aggressive compared with dormant tumors, and this inversely correlated with the levels of phosphorylated p38 and extracellular signal-regulated kinase1/2 (ERK1/2) MAPKs, two canonical MKP-1 targets. We demonstrate that MKP-1 protein levels are regulated by Notch3 in T-ALL cell lines because its silencing by RNA interference or treatment with γ-secretase inhibitors induced strong MKP-1 reduction whereas activation of Notch3 signalling had the opposite effect. Furthermore, MKP-1 has an important role in T-ALL cell survival because its attenuation by short hairpin RNA significantly increased cell death under stress conditions. This protective function has a key role in vivo, as MKP-1-deficient cells showed impaired tumorigenicity. These results elucidate a novel mechanism downstream of Notch3 that controls the survival of T-ALL cells.